The field of oxylipin research has outpaced the development of standards and guidelines. As these signaling lipids gain attention in inflammation, metabolism, and tissue repair, the field must confront a central challenge: many proposed pro-resolving mediators are difficult to measure and interpret. A recent publication by Schebb and colleagues calls for stronger evidence, from analytical detection to endogenous function.
Cholesterol exists in two forms in the human body: free and esterified as cholesteryl esters (CE). Free cholesterol is an essential component of cell membranes and participates in the biosynthesis of bile acids and steroid hormones. Cholesterol is transported as CE in lipoprotein particles and stored in cells as part of lipid droplets. Here, we present a rapid method (4 min) capable of analysing both cholesterol forms (free and esterified) in a single experiment, utilizing supercritical fluid chromatography (SFC) coupled with high-resolution mass spectrometry (QTOF). This method takes advantage of the commonly observed, yet often undesired, in-source fragmentation of cholesteryl esters during electrospray ionization (ESI). In addition, it offers the advantage of measuring the abundance of molecular CE species and it is suitable for low-volume samples as it can effectively analyse plasma or serum diluted up to 100-fold, requiring only a simple protein precipitation step for sample preparation. The method was validated using human plasma reference materials, showing excellent accuracy as demonstrated by the analysis of the NIST Standard Reference Material (SRM) 1950. Furthermore, the performances of this workflow for biomedical studies were tested in a large human Asian cohort for Coronary Heart Disease and in a cellular model of Niemann-Pick disease type C1 (NPC1), to follow separate pathways of cellular cholesterol metabolism.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulator of cholesterol homeostasis, primarily by promoting the lysosomal degradation of the hepatic low-density lipoprotein receptor (LDLR). However, emerging clinical and preclinical evidence suggests that PCSK9's influence extends beyond cholesterol clearance to the modulation of hepatic triglyceride metabolism. This review examines the biochemical mechanisms by which PCSK9 regulates intrahepatic triglyceride steady-state concentrations, highlighting its role in the assembly and lipidation of very low-density lipoproteins (VLDL) through both LDLR-dependent and independent mechanisms. We further discuss the integration of PCSK9 activity with hepatic lipid-sensing mechanisms and de novo lipogenesis. Finally, we evaluate the impact of current PCSK9-targeted therapies on the hepatic lipidome and contextualize these findings within the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD). By highlighting the non-canonical roles of PCSK9, we aim to critically assess its potential as a multifaceted therapeutic target for dyslipidemias and steatotic liver diseases.
Bone is a dynamic tissue that is continuously remodeled by tightly-coordinated actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Uncontrolled osteoclastogenesis has a severe impact on skeletal health and thus represents an important therapeutic target in osteolytic diseases. VprBP is a recently identified kinase and plays an important role in stimulating osteoclast differentiation and bone resorption through histone H2AT120 phosphorylation (H2AT120p). We previously developed B32B3 as the first VprBP inhibitor, but its high IC50 value has been a major challenge in using it for osteoclast-triggered bone diseases. In this study, we designed and characterized a series of small molecule compounds derived from B32B3 to identify a novel second-generation inhibitor of VprBP. Our initial screening of B32B3 derivatives identified three compounds, designated as B1486, B3703, and B6756, that are effective in interfering with VprBP kinase activity toward H2AT120p and RANKL-induced differentiation of osteoclast precursor cells (OCPs). However, when their cellular efficacy was systematically accessed over different concentration ranges, B1486 was more potent than B3703 and B6756 at hindering VprBP function in the process of OCP cell differentiation. Consistent with these in vitro findings, B1486 showed significantly improved inhibitory activity against VprBP-driven bone resorption and low bone mass phenotypes in our preclinical studies using zebrafish and mouse models. Taken together, our results indicate that B1486 is a highly potent inhibitor for blocking VprBP-mediated H2AT120p and osteoclastogenic gene silencing, as well as for overcoming osteoporosis generated by excessive osteoclast differentiation.
Natural killer (NK) cells are innate lymphocytes that directly eliminate tumor and virus-infected cells by integrating signals from activating and inhibitory receptors, and their effector functions are tightly coupled to cellular metabolism. Given that the inhibitory receptor PD-1 reprograms T cell metabolism to shape functional fate, the bioenergetic consequences of inhibitory receptor engagement on human NK cells remain largely unexplored, particularly for sialic acid-binding immunoglobulin-like lectin (Siglec-7), a glyco-immune checkpoint receptor. Here, we investigated metabolic programs and effector functions associated with Siglec-7 expression and antibody-mediated Siglec-7 ligation in primary NK cells and NK-92MI cells. Siglec-7POS NK cells exhibited selectively impaired CD107a degranulation under glycolytic and oxidative phosphorylation inhibition, whereas Siglec-7NEG cells remained relatively resistant, indicating distinct energetic wiring between these subsets. Engagement of Siglec-7 by an agonistic antibody induced mitochondrial fission with altered Drp1 phosphorylation, transient mitochondrial depolarization, and broadly suppressed mitochondrial respiration, while concurrently enhancing glycolytic capacity, consistent with a dual metabolic shift upon Siglec-7 ligation. In contrast, sustained Siglec-7 expression in NK-92MI-S cells was associated with globally enhanced mitochondrial respiratory capacity, indicating that sustained Siglec-7 expression and short-term treatment with an agonistic anti-Siglec-7 antibody were associated with distinct metabolic profiles in NK cells. Furthermore, Siglec-7POS NK cells showed increased accumulation of autophagic vacuole, reduced proliferation, and heightened apoptotic susceptibility compared with Siglec-7NEG counterparts. Collectively, these findings support an association between Siglec-7 status, mitochondrial homeostasis, and metabolic fitness in NK cells, with Siglec-7NEG cells retaining a metabolically robust, cytotoxic phenotype.
